Mammalian cell line expressing basement membrane proteins

Abstract

EHS murine tumor cells express basement membrane proteins such as laminin, entactin, collagen and proteoglycan. An immortal mammalian cell line designated BAM having phenotypic characteristics of EHS murine tumor cells may be prepared by culture of EHS cells in nutrient culture medium supplemented with low concentration of mammalian serum. BAM cells express basement membrane proteins in vitro.

What is claimed is: 
     
       1. A process for producing basement membrane proteins in vitro comprising growing immortalized EHS murine tumor cells in nutrient culture medium wherein said culture medium is Dulbecco's modification of Eagle's minimum essential medium supplemented with 1% to 5% mammalian serum, 3 to 7 μg/ml insulin, 3 to 7 μg/ml transferrin, 3 to 7 ng/ml sodium selenite, 300 to 400 ng/ml phosphoethanolamine, 100 to 200 ng/ml ethanolamine, 5 to 15 ng/ml epidermal growth factor, and 5 to 15 μg/ml gentamicin sulfate for a sufficient time to allow for deposit of basement membrane proteins in extracellular matrices; and isolating the resulting basement membrane proteins from the cells or cell product. 
     
     
       2. A method of propagating difficult cells comprising: a) growing immortalized EHS murine tumor cells in culture vessels containing nutrient culture medium supplemented with 1% to 5% mammalian serum, 3 to 7 μg/ml insulin, 3 to 7 μg/ml transferrin, 3 to 7 ng/ml sodium selenite, 300 to 400 ng/ml phosphoethanolamine, 100 to 200 ng/ml ethanolamine, 5 to 15 ng/ml epidermal growth factor, and 5 to 15 μg/ml gentamicin sulfate for a sufficient time for deposit of basement membrane proteins in extracellular matrices in said culture vessels;   b) removing said immortalized EHS murine tumor cells from said culture vessels and   c) adding difficult cells to said culture vessels in which basement membrane proteins have been deposited in step a).   
     
     
       3. A process for obtaining an immortalized basement membrane producing cell line which comprises incubating EHS tumor explants in nutrient culture medium supplemented with 1% to 5% mammalian serum, 3 to 7 μg/ml insulin, 3 to 7 μg/ml transferrin, 3 to 7 ng/ml sodium selenite, 300 to 400 ng/ml phosphoethanolamine, 100 to 200 ng/ml ethanolamine, 5 to 15 ng/ml epidermal growth factor, and 5 to 15 μg/ml gentamicin sulfate, for a sufficient time to reach confluency. 
     
     
       4. An immortalized EHS cell line which expresses laminin, collagen type IV, entactin and basement membrane specific heparan sulfate proteoglycan. 
     
     
       5. The immortalized EHS cell line of claim 4 wherein said laminin comprises laminin A, laminin B1 and laminin B2.